Exploring the Sequential Cellular Events of Phagocytosis Triggered by Godanti Bhasma in Mammalian Cells

Summary

This study elucidates phagocytic processing of Godanti Bhasma (GB) particles in mammalian cells, characterizing their cellular uptake, vacuole dynamics, acidification, and degradation. These findings not only advance our understanding of fundamental phagocytosis mechanisms but also establish GB as a promising model system for developing novel therapeutic strategies.

Abstract

Phagocytosis is a vital cellular mechanism through which cells engulf and degrade foreign particles, pathogens, or debris, playing a key role in immune defense and the maintenance of tissue homeostasis. Disruptions in this process are associated with various diseases. To explore the complex events involved in the phagocytosis pathway, advanced smart particles and effective monitoring techniques are essential. Godanti Bhasma (GB), a traditional Indian medicine composed of bioactive calcium sulfate particles, is rapidly internalized by phagocytosis in mammalian cells, inducing significant cytoplasmic vacuolation. The key stages of GB-induced phagocytosis were evaluated here using flow cytometry (FC), live-cell imaging, and specific staining techniques. Flow cytometric analysis demonstrated the formation of phagocytic cup-like structures associated with particle internalization. Live-cell imaging enabled real-time observation of phagocytic processes, including particle uptake, vacuole formation, degradation of engulfed materials, and vacuolar turnover. Staining with neutral red and acridine orange was employed to assess vacuolar acidification. Interestingly, treatment with the lysosomal inhibitor BFA1 in GB-treated cells did not lead to vacuolation, as evidenced by the lack of neutral red uptake, emphasizing the requirement for an acidic environment for vacuolation to occur. These findings underscore the potential of GB-induced phagocytosis as a model to elucidate the sequential cellular events involved in this process, which is critical for understanding host-pathogen interactions, intracellular trafficking, and developing innovative therapeutic strategies for disorders related to phagocytosis.

Introduction

Phagocytosis is an essential cellular process carried out by tissue-resident professional phagocytes, such as macrophages and dendritic cells, as well as non-professional phagocytes, such as epithelial cells and fibroblasts, which engulf and degrade foreign particles. These cells internalize and eliminate foreign particles, pathogens, or cellular debris, contributing significantly to immune defense and tissue homeostasis¹. This process begins with the recognition of pathogens, debris, or particles through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), which bind to pathogen-associated molecular patterns (PAMPs)². This recognition triggers the formation of a phagocytic cup, where plasma membrane protrusions extend around the target particle³. Actin filaments play a crucial role in this step, driving the membrane remodeling necessary for particle engulfment⁴. Once the particle is enclosed in a phagosome, a membrane-bound vesicle is formed. The phagosome undergoes maturation, characterized by the fusion with early, then late endosomes, followed by the incorporation of lysosomes containing hydrolytic enzymes⁵. These enzymes, such as cathepsins, lysozyme, and nucleases, are activated by acidification within the phagosome, achieved by vacuolar ATPase (V-ATPase), lowering the pH to 4.5-5.5. The degradative process concludes with the exocytosis of remnants from the cell. Dysfunctions in this dynamic and complex process can result in significant immune challenges^6,7,8.

To accurately evaluate the complex cellular process of phagocytosis, there is a need for improved and easy methods for monitoring the major cellular events of phagocytosis. Traditionally, monitoring method for phagocytosis quantification involve techniques such as fixing phagocytes at predetermined time intervals for microscopic visualization, measuring the internalization of dye-labeled targets through imaging or flow cytometry or counting the number of target cells remaining after a specified duration of phagocytosis, However, these methods do not capture the minute-to-minute fluctuations that occur during the phagocytosis process⁹. Moreover, methods relying on indirect indicators like dye uptake may not accurately reflect actual phagocytic events due to potential passive dye transfer¹⁰.

Recent advancements in live-cell imaging, flow cytometry, and staining techniques have revolutionized our ability to monitor phagocytosis with high spatiotemporal precision. Live-cell imaging, for instance, enables continuous observation of dynamic processes with sequential events tracking¹¹. Flow cytometry is widely used for analyzing mammalian cells and detecting biomarkers in clinical research¹². However, traditional FC methods are not equipped to evaluate the morphological and spatial characteristics of individual cells¹². Imaging Flow Cytometry (IFC) serves as a robust alternative that enables the collection of detailed information from single cells. IFC combines the analytical capabilities of flow cytometry with high-resolution imaging, allowing single-cell analysis with morphological and spatial detail. Various particles, including fluorescent latex beads, zymosan-APC, and synthetic nanoparticles, are commonly used to study phagocytosis, providing insights into receptor engagement, phagosome formation, and immune responses¹².

In contrast to conventional biochemical techniques, which typically rely on endpoint analysis, live-cell imaging provides continuous, dynamic monitoring of cellular processes. This method offers high spatiotemporal resolution and the ability to observe sequential events in phagocytosis. As a result, it is a powerful tool for studying the complex process of phagocytosis, allowing for more accurate and detailed insights into cellular behaviors and mechanisms¹³.

In addition to advancements in imaging systems, there is a need for smart phagocytic particles that can distinctly highlight phagocytic events, particularly the degradation steps occurring within the phagosome. Phagocytosis studies often utilize particles such as latex beads, zymosan, bacteria, apoptotic cells, and synthetic nanoparticles to explore mechanisms like receptor engagement, phagosome formation, and immune responses. While these particles provide valuable insights, they have limitations in tracking the complete degradation process or directly assessing phagosome maturation. For instance, latex beads are non-biodegradable and cannot demonstrate degradation, while zymosan and bacteria often require complex labeling and may not yield consistent degradation profiles^14,15.

Our previous study demonstrated the utility of GB, a traditional Ayurvedic formulation composed primarily of calcium sulfate, as an ideal particle for studying phagosome maturation and degradation¹⁶. GB dissolves over time within the acidic environment of the phagosome, allowing clear visualization of particle degradation. Furthermore, GB's ability to induce robust vacuole formation facilitates the investigation of distinct stages of phagosome maturation, from early formation to acidification and eventual resolution. Unlike other particles, GB eliminates the need for external labeling, providing a self-sustained system to study the complete lifecycle of phagosomes, making it an ideal model for understanding the dynamics of phagocytosis and associated disorders¹⁶.

This study presents a standardized and reproducible protocol designed to evaluate particle-induced phagocytosis and vacuole formation using Godanti Bhasma (GB) in both professional (RAW 264.7) and non-professional (HeLa and 3T3-L1) phagocytic cell lines. The overall goal of this method is to provide a robust platform for studying cellular uptake mechanisms, vacuolar biogenesis, and intracellular particle processing across different cell types. GB was suspended in DMEM at a concentration of 10 mg/mL, and to ensure uniformity, larger particles were allowed to settle while the upper fraction was collected for experimental use. For flow cytometry assays, cells were seeded at a density of 2 x 10⁵ cells per well in 12-well plates, and 300 µL of the GB working suspension was added to each well. For imaging-based analyses, 5,000-10,000 cells were seeded per well in 96-well plates or on chamber slides. This method establishes GB as a reliable model for investigating vacuole formation and particle internalization in diverse cellular types.

Protocol

1. Cell culture and seeding

1. Prepare complete DMEM medium supplemented with 10% heat-inactivated FBS and antibiotics (100 µg/mL penicillin, 100 µg/mL streptomycin). Sterilize all reagents and equipment before use.
2. Culture the 3T3-L1, HeLa, and RAW 264.7 cells in 25 cm² flasks and 35 mm culture dishes using DMEM. Maintain the cultures at 37 °C in a humidified incubator with 5% CO₂ and replace the medium every 48 h.
3. For subculturing, wash the cells 2x with PBS, then detach adherent cells using 500 µL of Trypsin-EDTA and incubate for 5 min. Add 1 mL of DMEM to neutralize the Trypsin-EDTA solution, pipette to create a single-cell suspension, and adjust the volume to 10 mL with fresh DMEM. Centrifuge at 300 x g for 5 min, resuspend the pellet in 1 mL of fresh medium.
4. Mix 10 µL of cell suspension with 0.4% trypan blue (1:1), count viable cells using a hemocytometer, calculate density as Cells/mL = Avg. count x 10⁴ x 2.
5. For 96-well plates: Dispense 100 µL of the cell suspension (containing 10,000 cells) into each well. For cavity slides, seed 50 µL of the cell suspension (containing 5000 cells) into two cavities, leaving the middle cavity empty. Add 50 µL of DMEM to each of the seeded cavities.
6. Incubate the plates and slides at 37 °C in a 5% CO₂ for 24 h to allow cell adherence and stabilization. Inspect the cells under an inverted microscope at 20x magnification to assess morphology, confluency, and contamination before proceeding with the experiments.

2. Preparation of GB stock solution/cocktail for cell culture

1. Procure GB and weigh 100 mg of the powder precisely. Suspend the powder in 10 mL of DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
2. Vortex the suspension and let it stand for 1 min to allow larger particles to settle. Then, transfer the upper 5 mL of the suspension into a sterile centrifuge tube.
3. Designate this 5 mL prepared GB suspension as the stock solution for subsequent cell culture experiments. Vortex the suspension thoroughly to ensure even dispersion of the particles before adding it to the cells.

3. Flow cytometry analysis

1. Harvest RAW 264.7, HeLa, or 3T3-L1 cells by trypsinization, neutralize with complete DMEM, centrifuge at 300 x g for 5 min, resuspend in 1 mL of fresh medium, mix 10 µL with 0.4% trypan blue (1:1), count viable cells using a hemocytometer, calculate density and adjust to 2 x 10⁵ cells/mL.
2. Seed 2 x 10⁵ cells per well in a 12-well plate and culture for 24 h. Incubate at 37 °C with 5% CO₂.
3. Treat the cells with GB suspension (300 µL per well) for various durations: 30 min, 1 h, and 12 h. After treatment, wash the cells 5x with PBS to remove excess particles.
4. Trypsinize the cells as in step 1.3 and wash them thoroughly with PBS. Load the prepared suspension into the sample port of the flow cytometer. Launch the software and select the appropriate acquisition settings. Choose Bright Field Mode for imaging.
5. Start data acquisition by clicking Run in the software. Acquire data for at least 10,000 events as the system captures images of individual cells passing through the flow cell.
6. Use area versus aspect ratio scatter plots to gate single intact cells. This helps exclude doublets and clusters. View the corresponding images in the image library. Collect individual cell images from the software image library to study the morphological changes induced by the treatment.
7. For particle analysis, seed 2 x 10⁵ cells per well in 12-well plates and culture for 24 h. Treat the cells with GB suspension (300 µL per well) for different durations: 1 h, 7 h, 14 h, and 24 h.
8. After treatment, gently shake the culture plate for 1 min and collect the culture media into 1.5 mL tubes. Wash the cells 5x with PBS to remove any extracellular particles.
9. Add 500 µL of RIPA buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1% Triton X-100; 1% sodium deoxycholate) to completely lyse the cells.
10. Run the culture medium and lysed cell samples containing particles separately through the flow cytometer. Load the samples into the sample port of the system. Launch the software and select the appropriate acquisition settings. Choose Bright Field Mode for particle analysis.
11. Start data acquisition by clicking Run in the software. Acquire data for at least 20,000 events as the system captures images of individual particles passing through the flow cell.
12. Use area versus aspect ratio scatter plots to gate all particles. Use the scatter plot tools to analyze particle count.

4. Time-lapse microscopy

1. Culture 3T3-L1 cells (3.0 x 10⁴) in a 35 mm dish with 2 mL of DMEM at 37 °C in a 5% CO₂ until they reach 70% confluency.
2. Thoroughly mix 300 µL of GB suspension into the pre-warmed fresh 2 mL culture medium and then place the culture dish with the cells and GB, on the imaging microscope stage.
3. Select the 10x Objective. Turn on the brightfield illumination using the Bright button. Click the Movie button to enter time-lapse mode. Set the interval time to 5 min. Start recording by clicking the Rec button.
4. Capture images at every 5 min intervals for 16 h to 24 h, ensuring continuous imaging under standard conditions in a CO₂ incubator.
5. Use ImageJ software to compile the captured images into a time-lapse video. This technique allows for the differentiation of various stages of phagocytosis, from internalization to complete degradation of particles.

5. Neutral red staining

1. After 24 h of cell growth in 96-well plates and cavity slides, thoroughly mix the GB particle suspension stock and add 30 µL of the suspension to each well or cavity.
2. Incubate at 37 °C with 5% CO₂ for 24 h to induce vacuole formation. Use untreated wells or cavities as negative controls.
3. For Neutral Red (NR) staining, prepare a 0.5 mg/mL NR solution in serum-free DMEM, and filter it through a 0.2 µm filter.
4. For imaging of vacuoles in 96-well plates and cavity slide: remove the media, add 60 µL of NR dye, and incubate at 37 °C for 15 min. Wash 3x with PBS to remove excess dye and then proceed with microscopic imaging.
5. Examine the vacuoles under a microscope using the 20x objective, to assess size, morphology, and number of vacuoles.

6. Treatment with Bafilomycin A1 for vacuole formation and acidification inhibition

1. Prepare a stock solution of Bafilomycin A1 (BFA1) at a concentration of 100 µg/mL (160.5 nM) by dissolving 100 µg of BFA in 50 µL of DMSO, followed by dilution in 950 µL of DMEM. Dilute 1 µL of this stock in 1.6 mL of DMEM to obtain a working solution of 0.1 nM.
2. Mix thoroughly and add 100 µL of the working BFA solution to wells containing cells in the 96-well plates. Also, add 30 µL of GB particle suspension to assess inhibition of vacuole and phagosome formation.
3. To inhibit phagosome acidification, add 100 µL of the working BFA solution to wells with pre-formed vacuoles (previously treated with GB particles). Incubate all wells at 37 °C with 5% CO₂ for 24 h.
4. Add 60 µL of Neutral Red (NR) dye to each well, incubate for an additional 15 min, and analyse vacuole formation and acidification.

7. Acridine orange (AO) staining

1. Treat the cells cultured in cavity slides with 50 µL of GB and incubate overnight under standard experimental conditions.
2. After treatment, incubate the cells with acridine orange solution (1 mg/mL) for 15 min at 37 °C to enable selective staining of vacuoles and acidic vesicular organelles.
3. Wash the cells 3x with sterile phosphate-buffered saline (PBS) to remove unbound dye and minimize background fluorescence.
4. Quickly analyze the slides with a fluorescence microscope equipped with a 20x objective and a blue excitation filter range of 450-492 nm, to assess cellular morphology and acridine orange uptake, which will provide insights into vacuolar dynamics.

Results

The study highlights key cellular responses to GB particles in mammalian cells. We used an imaging flow cytometer to investigate the internalization of GB particles in 3T3-L1 cells (Figure 1). The cells were exposed to particles for durations ranging from 30 min to 12 h. After 30 min, we observed the particles on the cell surface (Figure 1B), and the cell membrane began to fold inward at the attachment sites, indicating the initiation of internalization (

Discussion

Phagocytosis serves as a crucial immune defense mechanism, enabling the engulfment and subsequent elimination of particles, pathogens, and apoptotic cells. To understand the processes of phagosome formation and maturation, smart biocompatible particles combined with advanced imaging techniques are essential for studying dynamic events such as the vacuolar progression, acidification, and degradation of engulfed materials^18,19. We employed GB, a biocompatible model...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
25 cm2 Culture Flasks	NEST, India	707001
96- and 6-well plates	Tarsons India Pvt. Ltd., Kolkata, India; Thermo Scientific, India	980040; 140675
Acridine orange	SRL, India	82407
Antibiotics, and Trypsin-EDTA solution	High Media, India	628242; 655111
Cell Lines- HeLa, 3T3-L1 and RAW-264.7	National Centre For Cell Science (NCCS), Pune, Maharastra
culture dishes (35mm)	SPL Lifesciences, India	101350
Dulbecco’s modified Eagle medium (DMEM)	MP Biomedicles, India	1033126
Fetal bovine serum (FBS)	High Media, India	651257
Flow cytometer	Millipore, USA	Amnis	Amnis Imaging Flow Cytometers,
Fluorscent Microscope	DiGi Lab, India	Model: DiGi 110 Infinity
Godanti Bhasma	Patanjali Ayurved	240002
Neutral Red dye	CDH, India	593630
Time lapse microscope	NanoEnTek Inc.  Seoul, Korea.	JuLi	JuLi smart fluorescent cell analyzer,
Time lapse microscope	LEEDZ MICRO IMA. UK	S.No. LIM225552358A58	EA PRIME Inverted microscope